In the past 15 years or so, enormous progress has been made in the development of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides (RNA sequences) and their analogues, see'Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotides and Analogs', Agrawal, S. Ed., Humana Press, Totowa, 1993. Much of the work has been carried out on a micromolar or even smaller scale, and automated solid phase synthesis involving monomeric phosphoramidite building blocks Beaucage, S. L. ; Caruthers, M. H. Tetrahedron Lett., 1981,22,1859-1862 has proved to be the most convenient approach. Indeed, high molecular weight DNA and relatively high molecular weight RNA sequences can now be prepared routinely with commercially available synthesisers. These synthetic oligonucleotides have met a number of crucial needs in biology and biotechnology.

Following Zamecnik and Stephenson's seminal discovery that a synthetic oligonucleotide could selectively inhibit gene expression in Rous sarcoma virus, (Zamecnik, P.; Stephenson, M. Proc. Natl. Acad. Sci. USA 1978,75,280-284), the idea that synthetic oligonucleotides or their analogues might well find application in chemotherapy has attracted a great deal of attention both in academic and industrial laboratories. For example, the possible use of oligonucleotides and their phosphorothioate analogues in chemotherapy has been highlighted in the report of Gura, T. Science, 1995,270,575-577. The so-called antisense and antigene approaches to chemotherapy (Oligonucleotides. Antisense Inhibitors of Gene Expression, Cohen. J. S., Ed., Macmillan, Basingstoke 1989 Moser, H. E.; Dervan, P. B. Science 1987,238,645- 649), have profoundly affected the requirements for synthetic oligonucleotides. Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials. Several oligonucleotide analogues that are potential antisense drugs are now in advanced clinical trials. If, as seems likely in the very near future, one of these sequences becomes approved, say, for the treatment of AIDS or a form of cancer, kilogram or more probably multikilogram quantities of a specific sequence or sequences will be required.

Three main methods, namely the phosphotriester (Reese, Tetrahedron, 1978), phosphoramidite (Beaucage, S. L. in Methods in Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 33-61) and H-phosphonate (Froehler, B. C. in Methods in Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 63-80) approaches have proved to be effective for the chemical synthesis of oligonucleotides. While the phosphotriester approach has been used most widely for

synthesis in solution, the phosphoramidite and H-phosphonate approaches have been used almost exclusively in solid phase synthesis. The conventional H-phosphonate synthesis approach has been found to have a number of disadvantages. First, the process involves the use of an intermediate chain comprising a plurality of reactive H- phosphonate internucleotide linkages. The reactivity of these linkages can cause degradation, and hence lower yields and purities. Additionally, the use of a single oxidation or sulphurisation step at the end of the assembly of the desired molecule means that the process cannot readily be employed for the controlled production of chimeric nucleotides. Furthermore, when an oxidation step is employed, this is slow, and can cause concomitant degradation, and when a sulphurisation step is employed, not only are the reagents toxic, but the reaction is also slow. Additionally, the most common sulphurisation reagent, the so-called"Beaucage Reagent", can introduce a significant, and unpredictable, proportion of oxygen, in place of the expected sulphur. Attempts to improve the conventional H-phosphonate approach, for example that taught in EP-A-0 219 342 using acylating agents themselves have problems, for example the low coupling yields achieved with acylating agents.

According to the present invention, there is provided a process for the synthesis of a phosphorothioate triester comprising the reaction, in the presence of a coupling agent, of an H-phosphonate with a substrate comprising a free hydroxy group and bonded to a solid support, thereby forming a supported H-phosphonate diester, and subjecting the H- phosphonate diester to sulphur transfer with a sulphur transfer agent thereby forming a phosphorothioate triester. In many highly preferred embodiments, a plurality of coupling and sulphur transfer steps are carried out, with a sulphur transfer step being carried out after each coupling step.

The H-phosphonate employed in the process of the present invention is often an H-phosphonate monoester, and advantageously a protected nucleoside or oligonucleotide H-phosphonate, preferably comprising a 5'or a 3'H-phosphonate function, particularly preferably a 3'H-phosphonate function. Preferred nucleosides are 2'- deoxyribonucleosides and ribonucleosides ; preferred oligonucleotides are oligodeoxyribonucleotides and oligoribonucleotides. 2'-deoxyribonucleosides and oligodeoxyribonucleotides may comprise 2'-C-alkyl and 2'-C-alkenyl substituents.

When the H-phosphonate building block is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a 3' H-phosphonate function, the 5'hydroxy function is advantageously protected by a suitable protecting group. Examples of such suitable protecting groups include acid labile protecting groups, particularly trityl and substituted trityl groups such as dimethoxytrityl and 9-phenylxanthen-9-yl groups; and base labile-protecting groups such as FMOC.

Further protecting groups that may be employed include silyl ether groups.

When the H-phosphonate building block is a protected deoxyribonucleoside,

ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a 5' H-phosphonate function, the 3'hydroxy function is advantageously protected by a suitable protecting group. Suitable protecting groups include those disclosed above for the protection of the 5'hydroxy functions of 3'H-phosphonate building blocks and acyl, such as levulinoyl and substituted levulinoyl, groups.

When the H-phosphonate is a protected ribonucleoside or a protected oiigoribonucleotide, the 2'-hydroxy function is advantageously protected by a suitable protecting group, for example an acid-labile acetal protecting group, particularly 1- (2- fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp); and alkyl and aryl silyl protecting groups such as t-butyidiphenyl silyl groups, commonly trialkylsilyl groups, often tri (C, 4-alkyl) silyl groups such as a tertiary butyl dimethylsilyl group. Alternatively, the ribonucleoside or oligoribonucleotide may be a 2'-O-alkyl, 2'-O-alkoxyalkyl or 2'-O-alkenyl derivative, commonly a Cl. 4 alkyl, C14 alkoxyC, 4alkyl or alkenyl derivative, in which case, the 2' position does not need further protection.

Other H-phosphonates that may be employed in the process according to the present invention are derived from polyfunctional alcohols, especially alkyl alcools, and preferably diols or triols. Examples of alkyl diols include ethane-1,2-diol, and low molecular weight poly (ethylene glycols), such as those having a molecular weight of up to 400. Examples of alkyl triols include glycerol and butane triols. Further polyfunctonal alcools include saccharides, especially abasic nucleosides, for example ribose and deoxyribose. Commonly, only a single H-phosphonate function will be present, the remaining hydroxy groups being protected by suitable protecting groups, such as those disclosed hereinabove for the protection at the 5'or 2'positions of ribonucleosides.

The substrate comprising a free hydroxy group employed in the process of the present invention is commonly a protected nucleoside or oligonucleotide comprising a free hydroxy group, preferably a free 3'or 5'hydroxy group, and particularly preferably a 5'hydroxy group.

When the substrate comprising a free hydroxy group is a protected nucleoside or a protected oligonucleotide, preferred nucleosides are deoxyribonucleosides and ribonucleosides and preferred oligonucleotides are oligodeoxyribonucleotides and oligoribonucleotides.

When the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a free 5'-hydroxy group, the substrate comprising a free hydroxy group is preferably bonded to the solid support via the 3'-hydroxy function.

When the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a free 3'-hydroxy group, the substrate comprising a free hydroxy group is preferably bonded to the solid support via the 5'-hydroxy function.

When the substrate comprising a free hydroxy group is a ribonucleoside or an oligoribonucleotide, the 2'-hydroxy function is advantageously protected by a suitable protecting group, such as an acetal, particularly 1- (2-fluorophenyl)-4-methoxypiperidine-4- yl (Fpmp); and trialkylsilyl groups, often tri (C, 4-alkyl) silyl groups such as a tertiary butyl dimethyl silyl group. Alternatively, the ribonucleoside or oligoribonucleotide may be a 2'- O-alkyl, 2'-O-alkoxyalkyl or 2-'O-alkenyl derivative, commonly a C, 4 alkyl, C, 4 alkoxyC, 4alkyl or alkenyl derivative, in which case, the 2'position does not need further protection.

Other substrates comprising a free hydroxy group that may be employed in the process according to the present invention are saccharides, especially abasic nucleosides such as ribose and deoxyribose, and non-saccharide polyols, especially alkyl polyols, and preferably diols or triols. Examples of alkyl diols include ethane-1,2-diol, and low molecular weight poly (ethylene glycols), such as those having a molecular weight of up to 400. Examples of alkyl triols include glycerol and butane triols. Commonly, only a single free hydroxy group will be present, the remaining hydroxy groups being protected by suitable protecting groups, such as those disclosed hereinabove for the protection at the 5'or 2'positions of ribonucleosides, or being employed to bond the substrate to the solid support. However, more than one free hydroxy group may be present if it is desired to perform identical couplings on more than one hydroxy group.

In addition to the presence of hydroxy protecting groups, bases present in nucleosides/nucleotides employed in present invention are also preferably protected where necessary by suitable protecting groups. Protecting groups employed are those known in the art for protecting such bases. For example, adenine (A) and/or cytosine (C) can be protected by benzol, including substituted benzol, for example alkyl-or alkoxy-, often C, 4 alkyl-or C, 4alkoxy-, benzoyl ; pivaloyl ; and amidine, particularly dialkylaminomethylene, preferably di (C, 4-alkyl) aminomethylene such as dimethyl or dibutyl aminomethylene. Guanine (G) may be protected by a phenyl group, including substituted phenyl, for example 2,5-dichlorophenyl and also by an isobutyryl group. G may also be protected by diphenylcarbamoyl and glyoxal type protecting groups. thymine (T) and uracil (U) generally do not require protection, but in certain embodiments may advantageously be protected, for example at 04 by a phenyl group, including substituted phenyl, for example 2,4-dimethylphenyl or at N3 by a pivaloyloxymethyl, benzol, alkyl or alkoxy substituted benzol, such as C, 4 alkyl-or C14 alkoxybenzoyl.

After the coupling and sulphur transfer steps, and when it is desired to carry out further coupling and sulphur transfer steps, it is often necessary to introduce a free hydroxyl group into the phosphorothioate triester produced by the process of the present invention. When the phosphorothioate triester produced is a protected nucleoside or oligonucleotide having protected hydroxy groups, one of these protecting groups may be removed after carrying out the process of the first invention. Commonly, the protecting group removed is that on the 5'-hydroxy function. After the protecting group has been

removed, the oligonucleotide thus formed may then proceed through further stepwise or block coupling and sulphur transfers according to the process of the present invention in the synthesis of a desired oligonucleotide sequence. The method may then proceed with steps to remove the protecting groups from the internucleotide linkages, the 3'and the 5'- hydroxy groups and from the bases, and to cleave the product from the solid support.

The process according to the present invention may comprise a capping step, where hydroxy groups which are unreacted after a given coupling are capped to prevent further reaction in later couplings. Capping agents which may be employed are those known in the art for such a step, and include for example acylating agents such as acetic anhydride, (preferably in the presence of a nucleophilic acylation catalyst such as 4- (N, N- dimethyl) aminopyridine) and lower, eg up to C4, alkyl H-phosphonates, such as ethyl H- phosphonate, and 2-cyanoethyl H-phosphonate.

In a particularly preferred embodiment, the invention provides a method comprising the coupling of a 5'-O- (4, 4'-dimethoxytrityl)-2'-deoxyribonucleoside 3'-H- phosphonate or a protected oligodeoxyribonucleotide 3'-H-phosphonate and a substrate supported on a solid support with a free hydroxy function, most commonly a 2'- deoxyribonucleoside or oligodeoxyribonucleotide, in the presence of a suitable coupling agent and subsequent sulphur transfer in the presence of a suitable sulphur-transfer agent.

In the process of the present invention, any suitable coupling agents and sulphur- transfer agents available in the prior art may be used.

Examples of suitable coupling agents include alkyl and aryl acid chlorides, alkane and arene sulphonyl chlorides, alkyl and aryl chloroformates, alkyl and aryl chlorosulphites and alkyl and aryl phosphorochloridates, and carbodiimides.

Examples of suitable alkyl acid chlorides which may be employed include up to C12 alkyl acid chlorides, including adamantyl carbonyl chloride, and especially C2 to C7 alkanoyl chlorides, particularly pivaloyl chloride. Examples of aryl acid chlorides which may be employed include substituted and unsubstituted benzoyl chlorides, such as C, 4 alkoxy, halo, particularly fluoro, chloro and bromo, and Cl-, alkyl, substituted benzoyl chlorides. When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.

Examples of suitable alkanesulphonyl chlorides which may be employed include C2 to C7 alkanesulphonyl chlorides. Examples of arenesulphonyl chlorides which may be employed include substituted and unsubstituted benzenesulphonyl chlorides, such as C, 4 alkoxy, halo, particularly fluoro, chloro and bromo, and C14 alkyl, substituted benzenesulphonyl chlorides. When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.

Examples of suitable alkyl chloroformates which may be employed include C2 to C7 alkyl chloroformates. Examples of aryl chloroformates which may be employed include

substituted and unsubstituted phenyl chloroformates, such as C, 4 alkoxy, halo, particularly fluoro, chloro and bromo, and C, 4 alkyl, substituted phenyl chloroformates.

When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.

Examples of suitable alkyl chlorosulphites which may be employed include C2 to C7 alkyl chlorosulphites. Examples of aryl chlorosulphites which may be employed include substituted and unsubstituted phenyl chlorosulphites, such as C, alkoxy, halo, particularly fluoro, chloro and bromo, and C,-, alkyl, substituted phenyl chlorosulphites.

When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.

Examples of suitable alkyl phosphorochloridates which may be employed include di (C, to C6 alkyl) phosphorochloridates. Examples of aryl phosphorochloridates which may be employed include substituted and unsubstituted diphenyl phosphorochloridates, such as C14 alkoxy, halo, particularly fluoro, chloro and bromo, and C14 alkyl, substituted diphenyl phosphorochloridates. When substituted, from 1 to 5 substituents on each phenyl group may be present, particularly in the case of alkyl and halo substituents.

Further coupling agents that may be employed are the chloro-, bromo-and (benzotriazo-1-yloxy)-phosphonium and carbonium compounds disclosed by Wada et al, in J. A. C. S. 1997,119, pp 12710-12721 (incorporated herein by reference).

Examples of suitable carbodiimides that may be employed include alkyl carbodiimides, especially (C, to C6 alkyl) carbodiimides, such as 1,3 dicyclohexyl carbodiimide, 1,3 diisopropyl carbodiimide, 1-tert.-butyl-3-ethyl carbodiimide and 1- (3- dimethylaminopropyl)-3-ethyl carbodiimide. One or more of the alkyl groups may be substituted, for example by an alkyl amino moiety. An example of a substituted alkyl carbodiimide is 1- (3-dimethylaminopropyl)-3-ethyl carbodiimide.

Preferred coupling agents are diaryl phosphorochloridates, particularly those having the formula (ArO) 2POCI wherein Ar is preferably phenyl, 2-chlorophenyl, 2,4,6- trichlorophenyl or 2,4,6-tribromophenyl.

The sulphur transfer agents employed in the process of the present invention introduce a protected thio moiety into the linkage, thereby forming a phosphorothioate triester. The phosphorthioate triesters are commonly subsequently converted to phosphodiester or phosphorothioate diesters, and the sulphur transfer agent can be selected accordingly. For example. the nature of the sulphur-transfer agent will depend on whether an oligonucleotide, a phosphorothioate analogue or a mixed oligonucleotide/oligonucleotide phosphorothioate is required. Sulphur transfer agents employed in the process of the present invention often have the general chemical formula : ______p

wherein L represents a leaving group, and D represents an aryl group, a methyl or a substituted alkyl group or an alkenyl group. Commonly the leaving group is selected so as to comprise a nitrogen-sulphur bond. Examples of suitable leaving groups include morpholines such as morpholine-3, 5-dione; imides such as phthalimides, succinimides and maleimides ; indazoles, particularly indazoles with electron-withdrawing substituents such as 4-nitroindazoles ; and triazoles.

Where a standard phosphodiester linkage is required in the final product, the sulphur transfer agent is commonly selected such that the moiety D represents an aryl group, such as a phenyl or naphthyl group. Examples of suitable aryl groups include substituted and unsubstituted phenyl groups, particularly halophenyl and alkylphenyl groups, especially 4-halophenyl and 4-alkylphenyl, commonly 4-(C, 4 alkyl) phenyl groups, most preferably 4-chlorophenyl and p-tolyl groups. An example of a suitable class of standard phosphodiester-directing sulphur-transfer agent is an N- (arylsulphanyl) phthalimide (succinimide or other imide may also be used).

Where a phosphorothioate diester linkage is required in the final product, the moiety D commonly represents a methyl, substituted alkyl or alkenyl group. Examples of suitable substituted alkyl groups include substituted methyl groups, particularly benzyl and substituted benzyl groups, such as alkyl-, commonly C14alkyl-and halo-, commonly chloro-, substituted benzyl groups, and substituted ethyl groups, especially ethyl groups substituted at the 2-position with an electron-withdrawing substituent such as 2- (4- nitrophenyl) ethyl and 2-cyanoethyl groups. Examples of suitable alkenyl groups are allyl, propargyl and crotyl. Examples of a suitable class of phosphorothioate-directing sulphur- transfer agents are, for example, (2-cyanoethyl) sulphanyl derivatives such as 4- [ (2- cyanoethyl)-sulphanyl] morpholine-3, 5-dione or a corresponding reagent such as 3- (phthalimidosulphanyl) propanonitrile.

A suitable temperature for carrying out the coupling reaction and sulphur transfer is in the range of from approximately-55°C to about 40°C, such as from 0 to 30°C, and preferably about room temperature (commonly in the range of from 10 to 25°C, for example approximately 20°C).

The coupling and sulphur transfer steps of the process according to the present invention are carried out as often as is necessary to synthesise the desired number of phosphorothioate linkages.

Preferred nucleoside or nucleotide H-phosphonates employed in the process of the present invention have the general chemical formula:

wherein each B independently is an organic base; each Q independently is H, CH2R'or OR'wherein R'is alkyl, substituted alkyl, alkenyl or a protecting group; each R independently is an aryl, methyl, substituted alkyl or alkenyl group; W is H, a protecting group or an H-phosphonate group of formula in which M+ is a monovalent cation; each X independently represent O or S; each Y independently represents O or S; Z is H, a protecting group or an H-phosphonate group of formula in which M+ is a monovalent cation; and n is zero or a positive integer; provided that when W is H or a protecting group, that Z is an H-phosphonate group, and that when Z is H or a protecting group, that W is an H-phosphonate group.

Preferably, only one of W or Z is an H-phosphonate group, commonly only Z being an H-phosphonate group.

When W or Z represents a protecting group, the protecting group may be one of

those disclosed above for protecting the 3'or 5'positions respectively. When W is a protecting group, the protecting group is preferably a trityl group, particularly a dimethoxytrityl group. When Z is a protecting group, the protecting group is preferably a trityl group, particularly a dimethoxytrityl group, or an acyl group, preferably a levulinoyl group.

Organic bases which may be represented by B include nucleobases, such as natural and unnatural nucleobases, and especially purines, such as hypoxanthine, and particularly A and G, and pyrimidines, particularly T, C and U. The bases may be protected, with A, G and C preferably being protected. Suitable protecting groups include those described hereinabove for the protection of bases.

When Q represents a group of OR', and R'is alkenyl, the alkenyl group is often a C, 4 alkenyl group, especially allyl, propargyl or crotyl group. When R'represents alkyl, the alkyl is preferably a C14 alkyl group. When R'represents substituted alkyl, the substituted alkyl group includes alkoxyalkyl groups, especially C, 4 alkyoxyC, 4 alkyl groups such as methoxyethyl groups. When R'represents a protecting group, the protecting group is commonly an acid-labile acetal protecting group, particularly 1- (2- fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp) or a trialkylsilyl groups, often a tri (C, 4- alkyl) silyl group such as a tertiary butyl dimethylsilyl group.

Preferably, X represents O.

In many embodiments, Y represents S and each R independently represents methyl, substituted alkyl, alkenyl or aryl. Preferably, each R independently represents a methyl group; a substituted methyl group, particularly a benzyl or substituted benzyl group, such as an alkyl-, commonly C, 4alkyl-or halo-, commonly chloro-, substituted benzyl group; a substituted ethyl group, especially an ethyl group substituted at the 2- position with an electron-withdrawing substituent such as a 2- (4-nitrophenyl) ethyl or a 2- cyanoethyl group; a C, 4 alkenyl group, preferably an allyl and crotyl group; or a substituted or unsubstituted phenyl group, particularly a halophenyl or alkylphenyl group, especially 4-halophenyl group or a 4-alkylphenyl, commonly a 4- (Cl-4 alkyl) phenyl group, and most preferably a 4-chlorophenyl or a p-tolyl group.

M+ preferably represents a trialkyl ammonium ion, such as a tri (Ci4- alkylammonium) ion, and preferably a triethylammonium ion or a cation of a cyclic base such as 1,5-diazabicylo [4.3.0] non-5-ene (DBN) or 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU). n may be 0 or 1 up to any number convenient for the synthesis of the desired oligonucleotide, particularly up to about 20. Preferably n is 0 to 9, and especially 0 to 7.

H-phosphonates wherein n represents 1,2 or 3 can be employed when it is desired to add small blocks of nucleotide, with correspondingly larger values of n, for example 4,5, or 6, being employed if larger blocks of oligonucleotide are desired to be coupled.

The H-phosphonates, coupling agents and sulphur transfer agents can employed

as a solution, although the coupling agent or sulphur transfer agent may employed as a neat liquid or solid as appropriate. Organic solvents which can be employed include haloalkanes, particularly dichloromethane, esters, particularly alkyl esters such as ethyl acetate, and methyl or ethyl propionate, nitriles, such as acetonitrile, amides, such as dimethylformamide and N-methylpyrollidinone, and basic, nucleophilic solvents such as pyridine. Preferred solvents are pyridine, dichloromethane, dimethylformamide, N- methylpyrollidinone and mixtures thereof.

Protecting groups can be removed using methods known in the art for the particular protecting group and function. For example, transient protecting groups, particularly gamma keto acids such as levulinoyl-type protecting groups, can be removed by treatment with hydrazine, for example, buffered hydrazine, such as the treatment with hydrazine under very mild conditions disclosed by van Boom. J. H.; Burgers, P. M. J.

Tetrahedron Lett., 1976,4875-4878. The resulting partially-protected oligonucleotides with free 3'-hydroxy functions may then be converted into the corresponding H- phosphonates which are intermediates which can be employed for the block synthesis of oligonucleotides and their phosphorothioate analogues.

When deprotecting the desired product once this has been produced, protecting groups on the phosphorus which produce phosphorothioate triester linkages are commonly removed first. For example, a cyanoethyl group can be removed by treatment with anhydrous, strongly basic amine such as DABCO, 1,5-diazabicylo [4.3.0] non-5-ene (DBN), 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) or triethylamine.

Phenyl and substituted phenyl groups on the phosphorothioate internucleotide linkages and on the base residues can be removed by oximate treatment, for example with the conjugate base of an aldoxime, preferably that of E-2-nitrobenzaldoxime or pyridine-2-carboxaldoxime (Reese et al, Nucleic Acids Res. 1981). Kamimura, T. et al in J. Am. Chem. Soc., 1984,106 4552-4557 and Sekine, M. Et al, Tetrahedron, 1985,41, 5279-5288 in an approach to oligonucleotide synthesis by the phosphotriester approach in solution, based on S-phenyl phosphorothioate intermediates; and van Boom and his co-workers in an approach to oligonucleotide synthesis, based on S- (4-methylphenyl) phosphorothioate intermediates (Wreesman, C. T. J. et al, Tetrahedron Lett., 1985,26, 933-936) have all demonstrated that unblocking S-phenylphosphorothioates with oximate ions (using the method of Reese et al., 1978 ; Reese, C. B, ; Zard, L. Nucleic Acids Res., 1981,9,4611-4626) led to natural phosphodiester internucleotide linkages. In the present invention, the unblocking of S- (4-chlorophenyl)-protected phosphorothioates with the conjugate base of E-2-nitrobenzaldoxime proceeds smoothly and with no detectable internucleotide cleavage.

Other protecting groups, for example benzoyl, pivaloyl and amidine groups can be removed by treatment with concentrated aqueous ammonia.

Trityl groups present can be removed by treatment with acid. With regard to the

overall unblocking strategy in oligonucleotide synthesis, another important consideration of the present invention, is that the removal of trityl, often a 5'-terminal DMTr, protecting group ('detritylation') should proceed without concomitant depurination, especially of any 6-N-acyl-2'-deoxyadenosine residues.

Silyl protecting groups may be removed by fluoride treatment, for example with a solution of an ammonium fluoride, for example a solution of trialkylamine trihydrogen fluoride or a solution of a tetraalkyl ammonium fluoride salt such as tetrabutyl ammonium fluoride.

Fpmp protecting groups may be removed by acidic hydrolysis under mild conditions.

This new approach to the synthesis of oligonucleotides is suitable for the preparation of sequences with (a) solely phosphodiester, (b) solely phosphorothioate diester and (c) a combination of both phosphodiester and phosphorothioate diester internucleotide linkages.

Solid supports that are employed in the process according to the present invention are substantially insoluble in the solvent employed, and include those supports well known in the art for the solid phase synthesis of oligonucleotides. Examples include silica, controlled pore glass, polystyrene, copolymers comprising polystyrene such as polystyrene-poly (ethylene glycol) copolymers and polymers such as polyvinylacetate.

Additionally, poly (acrylamide) supports, especially microporous or soft gel supports, such as those more commonly employed for the solid phase synthesis of peptides may be employed if desired. Preferred poly (acrylamide) supports are amine-functionalised supports, especially those derived from supports prepared by copolymerisation of acryloyl-sarcosine methyl ester, N, N-dimethylacrylamide and bis-acryloylethylenediamine, such as the commercially available (Polymer Laboratories) support sold under the catalogue name PL-DMA. The procedure for preparation of the supports has been described by Atherton, E. ; Sheppard, R. C.; in Solid Phase Synthesis : A Practical Approach, Publ., IRL Press at Oxford University Press (1984). The functional group on such supports is a methyl ester and this is initially converted to a primary amine functionality by reaction with an alkyl diamine, such as ethylene diamine.

The substrate is commonly bound to the solid support via a cleavable linker.

Examples of linkers that may be employed include those well known in the art for the solid phase synthesis of oligonucleotides, such as urethane, oxalyl, succinyl, and amino- derived linkers.

In many embodiments when the substrate is bound to a poly (acrylamide) support via a cleavable linker and comprises a nucleoside, the substrate is attached to the support by a process comprising either: a) reacting a 5'-protected nuceloside having a free 3'-hydroxy group with a linker, preferably succinic anhydride, to form a linker-derivatised nucleoside ; and

b) reacting the linker-derivatised nucleoside with an amine-functionalised poly (acrylamide) support in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example diisopropylethylamine (DIPEA) or N-methylmorpholine (NMM), or hydroxybenzotriazole ; or c) reacting an amine-functionalised poly (acrylamide) support with a linker, preferably succinic anhydride, to form a linker-derivatised support; and d) reacting the linker-derivatised support with a 5'-protected nuceloside having a free 3'- hydroxy group in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example DIPEA or NMM, or hydroxybenzotriazole ; then in either case, removing the 5'-protecting group, which is preferably a trityl or substituted trityl group. However, it will be recognised that it may be desired to retain the 5'-protecting group in which case its removal may be omitted. In this case, the 5'- protecting group can be removed when desired prior to use of the supported substrate in the process for the synthesis of phosphorothioate triesters according to the present invention.

Coupling agents used for amide bond formation that can be employed in the process for attaching the substrate to an amine-functionalised poly (acrylamide) support include those known in the art of peptide synthesis, see for example those coupling reagents disclosed by Wellings, D. A.; Atherton, E.; in Methods in Enzymology, Publ., Academic Press, New York (1997) incorporated herein by reference, such as those comprising carbodiimides, especially dialkyl carbodiimides such as N, N'- diisopropylcarbodiimide (DIC), and reagents that form active esters, particularly benzotriazole active esters in situ, such as 2- (1H-benzotriazole-1-yl)-1, 1,3,3- tetramethyluronium tetrafluoroborate (TBTU) or benzotriazole-1-yloxy-tris- (dimethylamino) phosphonium hexafluorophosphate (BOP).

An organic solvent such as N, N-dimethylformamide (DMF) or N- methylpyrrolidinone (NMP) is suitably employed for attaching the substrate to an amine- functionalised poly (acrylamide) support.

The process for the synthesis of phosphorothioate triesters according to the present invention can be carried out by stirring a slurry of the substrate bonded to the solid in a solution of the H-phosphonate and coupling agent or sulphur-transfer agent.

Alternatively, the solid support can be packed into a column, and solutions of H- phosphonate and coupling agent, followed by sulphur transfer agent can be passed sequentially through the column.

The process according to the present invention is preferably employed to produce oligonucleotides typically comprising 3 or more bases. The upper limit will depend on the length of the oligonucleotide it is desired to prepare. Often, oligonucleotides produced by the process of the present invention comprise up to 40 bases, commonly up to 30 bases,

and preferably from 5 to 25, such as from 8 to 20, bases. The coupling and sulphur transfer steps of the process of the present invention are repeated sufficient times to produce the desired length and sequence.

On completion of the assembly of the desired product, the product may be cleaved from the solid support, preferably following deprotection of the product. When the desired product is an oligonucleotide, it will be recognised that the product will be a phosphate diester, phosphorothioate diester or chimera comprising both phosphate diester and phosphorothioate diester moieties. Cleavage methods employed are those known in the art for the given solid support. When the product is bound to the solid support via a cleavable linker, cleavage methods appropriate for the linker are employed.

Following cleavage, the product can be purified using techniques known in the art, such as one or more of ion-exchange chromatography, reverse phase chromatography, and precipitation from an appropriate solvent. Further processing of the product by for example ultrafiltration may also be employed.

The invention will now be illustrated without limitation by the following examples.

Example 1 4-N-benzoyl-5'-O- (4, 4'-dimethoxytrityl)-2'-deoxycytidine (DMT CbZ) supported via a succinimide linker at the 3'-position to a polystyrene support (commercially available under the trade name Pharmacia Primer Support 30HL, loading 84 umol/g ; 2g) was poured into a sintered vessel, wetted with 100ml of CH2CH2, and aerated with house nitrogen. The solvent was removed. Following this wash procedure, the supported DMT CbZ was treated as follows: i) with 100ml of 3% v/v dichloroacetic acid in dichloromethane (DCA/DCM)-Wait for 60 seconds, remove DCA/DCM ii) with 100ml of CH2CH2-Wait for 60 seconds, remove CH2CH2 iii) with 100ml of 3% DCA/DCM-Wait for 60 seconds, remove acid iv) Wash with 100moi CH2CH2-Wait for 60 seconds ; and finally v) Wash with more CH2CH2 (100ml) and dry resin with N2 to produce supported 4-N-benzoyl-2'-deoxycytidine (HOCbZ-poly) Quantities Equivs Amount 822 DMTAbZ (H) 5.0 0.84mmol-690mg HOCbZ-poly 1.0 0. 168mmol 232 CESP 10.0 1.68mmol-389mg 268.5/1/3 Activator* 10.0 1.68mol-0.35ml * As a 1 : 1 solution in DCM.-. use 0.69ml

6-N-benzoyl-5'-0- (4, 4'-dimethoxytrityl)-2'-deoxyadenosine-3'-H-phosphonate (DMTAbZ (H), 690mg) was dried by co-evaporation (2x5ml) with anhydrous pyridine, and combined with 2g of HOCbZ-poly (in a 100m1 florentine). Pyridine was added such that all the DMTAbZ (H) was in solution and a heterogeneous slurry was formed. This required 40ml of pyridine. The mixture was cooled to-40°C and this was maintained during both coupling and sulphur transfer stages. The reaction mixture was purged with argon, and the reaction was carried out under a blanket of this gas. 0.35ml (0.69ml of 1: 1 vol/vol mixture in anhydrous dichloromethane) of diphenylphosphorochloridate was added drop- wise over 5 minutes to the cooled, stirred slurry and left stirring for a further 15 minutes.

At this point 389mg 2-(2-cyanoethyl) sulphanyl phthalimide (CESP) in 8ml of pyridine was added in one aliquot. The mixture was initially stirred at-40°C and then allowed to warm to room temperature. The slurry was then transferred to a sinter, washed with pyridine (50mut) and DCM (200ml) and the resin dried under vacuum to produce 5'-(DMT)-AbZ-O- P (=O) (SCH, CH, CN)-C"- (3'-O-polymer support) ("DMT-AC-poly").

DMT-AC-poly (1.4g@71 pmolg~'loading) was treated for 60 seconds at room temperature with 200moi of a 3% solution of dichloroacetic acid in CH2CH2. The resin was washed with clean CH2CH2 and dried in a stream of N2 to produce 5'-(HO)-AbZ-O- P (=O) (SCH2CH2CN)-C"- (3'-O-polymer support) ("HO-AC-poly").

Equivs Amount 2235 DMTACAC (H) 5.0 0.46mol-1.03g 71 lmol/g HO-AC-poly 1.0 13g@ 71 lmol/g 232 CESP 10.0 0.923mmol-214mg 268.5/13 Activator 10.0 *0.923mmol-0.19ml *1/1 in CH2CH2.. 0.38ml A 5'-DMT protected nucleotide 3'-H-phosphonate having the base sequence 5'- DMT-ACAC, with each internucleotide being protected by a beta-cyanoethylthio moiety (5'-DMT-ACAC-3'-H phosphonate) was prepared from the corresponding tetrameric oligonucleotide comprising a free 3'-hydroxy group (5'-DMT-ACAC-3'-OH) as follows.

Ammonium toluyl-H-phosphonate was dissolve in 50ml methanol and 5ml triethylamine.

This mixture is evaporated to form a gum, and the gum redissolved in 100m1 pyridine, together with 12.3g 5'-DMT-ACAC-3'-OH, and the pyridine evaporated. The residue was then redissolved in 50m1 pyridine. The pyridine solution is cooled to-30°C and pivaloyl chloride (2.2ml) added over 1 minute. The mixture was stirred for 30 minutes, and 15ml water is added. After 10 minutes stirring, 250ml of a solution of 10% v/v methanol in dichloromethane was added, and this was washed with 0.5M triethylammonium phosphate buffer. The organic layer was separated, diluted with 25ml methanol and the

wash repeated. The organic layer was separated, evaporated from 100ml toluene, and then evaporated from 200ml 50: 50 by vol toluene/pyridine mixture. The crude gum is re- dissolve in dichloromethane, and purified by column chromatography to yield 5'-DMT- ACAC-3'-H phosphonate.

The 5'-DMT-ACAC-3'-H phosphonate was dried by co-evaporation from anhydrous pyridine (2x5ml) and re-suspended in 40ml of the same solvent. The mixture was combined with 1.3g of HO-AC-poly in a 100ml florentine. The heterogeneous mixture was stirred, blanketed in Argon and cooled to-40°C. 0.38ml of 1: 1 vol/vol diphenylphosphorochloridate in CH2CH2 was added drop-wise over 5 minutes, and the mixture stirred for a further 15 minutes. At this point CESP (214mg in 5ml pyridine) was added in one aliquot. The mixture was initially stirred at-40°C and then allowed to warm to room temperature over 20 minutes prior to quench (1 ml of water).

The slurry was transferred to a sinter, and was filtered and washed (5x100ml of CH2CH2) and dried over nitrogen to produce 5'-(DMT)-AbZ-O-P (=O) (SCH2CH2CN)-CbZ-O- P (=O) (SCH2CH2CN)-AbZ-O-P (=O) (SCH2CH2CN)-CbZ-O-P (=O) (SCH2CH2CN)-AbZ-O- P (=O) (SCH2CH2CN)-CbZ- (3'-O-polymer support) ("DMT-ACACAC-poly").

The cyanoethyl groups can be removed by treatment with anhydrous 1, 8- diazabicyclo [5,4,0] undec-7-ene to yield phosphorothioate linkages, and the phosphorothioate oligonucleotide could be cleaved from the support by treatment with concentrated aqueous ammonia containing 10% vol mercaptoethanol.

Example 2. General Methodology for H-Phosphonate Coupling and Sulfur Transfer on Poly (dimethylacrylamide) (PDMA) A poly (acrylamide) resin produced by copolymerisation of acryloyl-sarcosine methyl ester, N, N-dimethylacrylamide and bis-acryloylethylenediamine (PDMA resin, 69g) was treated with ethylene diamine (700moi) in a 2L round bottomed flask which was sealed and allowed to stand at room temperature overnight. The slurry was then transferred to a sinter funnel and washed with DMF (12x 700ml). This produced DMF washings containing no trace of amine. The resin was then washed with DMF containing an increasing gradient of DCM (2.5L ; 0-100% DCM) then an increasing gradient of ether in DCM (900mol ; 0-100% ether). The resin was then dried overnight in a stream of nitrogen at 40°C. The resin produced had an amino functionalisation of 973 micromoles per gram ("Amino-PDMA resin").

A solution of 4-N-benzoyl-5'-O- (4, 4'-dimethoxytrityl)-2'-deoxycytidine-3'-O- succinate ("DMT-CbZ-succinate", 3eqvs, 234 mmol), hydroxybenzotriazole (6 eqvs, 467 mmol) and DIC (diisopropyl carbodiimide; 4 eqvs, 311 mmol) was prepared in 1300ml of DMF. The solution was prepared in a 2L flask which had previously been silanised (this was achieved by simply swirling trimethylsilyl chloride around the flask) in order to prevent

the resin from adhering to glass. The Amino-PDMA resin (1 eqv, 78 mmol) was then added to the solution and left to stand overnight at room temperature. This resulted in a stiff non-mobile gel. After 22 hrs a few beads were removed and washed with fresh DMF. The resin was found to be Kaiser negative, indicating that all the amino groups on the resin had reacted with DMT-CbZ-succinate. The reaction mixture was then transferred to a sintered funnel (18cm diameter, porosity 3) and washed (5 x 800moi). After the final DMF wash a similar diethyl ether treatment as described for the amino PDMA was carried out in order to shrink the resin. After blowing dry with N2 for 24 hours and then drying in the vacuum oven overnight the resin was weighed. This gave a weight increase of 55.7g from the original 80g of Amino-PDMA. This resulted in DMT-CbZ-PDMA resin loaded to 573 mmol per gramme.

A 5'-DMT-deoxycytidine dimer (protected on the internucleoside phsophorus by a P-cyanoethyl group) was also attached to the resin at a similar loading using identical conditions to above.

The loaded resin was prepared for coupling by having the DMT group removed as follows. DMT-CbZ-PDMA resin is poured into a sintered funnel (7cm, porosity 3) and a positive pressure of nitrogen is applied. A 3% solution of DCA in DCM (15ml/g resin) is then added to the resin and left to bubble gently for 5 minutes. This was repeated (twice, quantities same as above). All of the orange colour was removed from the resin at this point indicating that detritylation is complete. Residual acid was removed by washing with DCM and further washing carried out with ether in DCM, starting at 20% v/v ether and continuing to 100% ether. The HO-CbZ-PDMA resin is then air dried and dried in-vacuo at 40°C overnight.

HO-CbZ-PDMA resin (1 mmol ; 1.48g), DMT-Cbz-H-phosphonate (6 eq; 4.79g) were weighed into a 100mut florentine. This was then suspended in 60ml of anhydrous grade DMF. The mixture was left for 5 minutes to allow the resin to swell to its maximum capacity at room temperature and pyridine (4.36ml) was added. The reaction mixture was then stirred using a small (12x5mm) flea (at maximum stir rate on a Heidolph MR3001 K stirrer hotplate). The neat activator, diphenyl phosphorochloridate (6 eq ; 1.24moi) was added dropwise, during a 5 minute period, using the syringe pump (Razel A99FZ). Immediately after the addition was complete, the sulfur transfer reagent (CESP : 232mg; 1 mmol), was added to the mixture as a solid single aliquot. After 5 minutes more the resin mixture was poured into a sintered funnel (porosity 3). The resin was then washed for 5 minutes, with stirring, using 50ml of DMF. This process was repeated a further two times. At this point any un-reacted hydroxyl groups were capped by treatment with 35ml of a 1.4M solution of acetic anhydride in pyridine containing 4- (N, N- dimethyl) aminopyridine (DMAP, 0.5mmol) for 20 mins, followed by further washing with DMF (3x 30ml). At the end of the final wash as much DMF as possible was removed prior to washing the resin with 100ml of diethyl ether. The resin was stirred during the ether addition and the solvent allowed to drop through the sinter under gravity. The ether

wash was repeated twice more. After the final wash the remaining ether was removed by passing N2 through the resin for 30 minutes. The resin was then dried overnight in vacuo at 40°C. The weight increase of the resin and trityl assay indicated quantitative coupling to form DMT-CbZCbZ_PpMA, protected on the internucleoside linkage with a ß-cyanoethyl group.

Preparation of 5'-HO-AbZCbZAbZCbZCbZ_PDMA HO-CbZ-PDMA resin prepared as above (1mmol ; 1.48g) and 5'-DMT-AbZCbZAbZCbZ- H-phosphonate protected on the internucleoside linkages with ß-cyanoethyl groups ( 1.2eq ; 2.68g) were weighed into a 100ml florentine. Anhydrous DMF (60ml) was added and the resin allowed to swell for 5 mins at room temperature. Pyridine (5mmol ; 0.4ml) was added followed by the coupling agent, diphenyl phosphorochloridite (2mmol ; 0.41ml) which was added dropwise during 5 mins using a syringe pump as above. On completion of this addition, sulfur transfer agent (CESP; 2.5mmol; 0.58g) was added solid as a single aliquot and the suspension stirred for one hour. The solid was then washed with DMF followed by DCM and the DMT group removed by treatment with dichloroacetic acid as above. The resin was then washed with DCM, DMF and finally ether before being dried in a stream of nitrogen and then in vacuo at 40°C overnight. The weight increase and a trityl assay indicated a yield of 75%.